The aim of this proposal is to develop an effective and rapid method for disrupting the poultry-to- human transmission of avian influenza by in ovo injection of flu vaccines that can be mass- administered with a mechanized injector. The hypothesis is that mass-vaccination of poultry against an outbreak of avian influenza reduces the dissemination of the virus to new flocks and consequently the risk to humans. As avian influenza gets more and more deadly, it is increasingly urgent to produce vaccines rapidly in response to an unprecedented escalation in demand. Emerging evidence suggests that poultry can be immunized en masse by in ovo administration of DMA vaccines and non-replicating adenovirus (Ad)-vectored vaccines without masking infections. Both vaccines are produced by molecular biology techniques without the requirement to propagate lethal virus strains. These studies will compare these two vaccination modalities in their potency to mobilize the immune repertoire toward a beneficial immune protection against avian influenza virus infection in chickens. In this project, efficacy of a DMA vaccine will be compared to that induced by a non-replicating Ad vector encoding the same codon-optimized A/Turkey/Wisconsin/68 hemagglutinin gene. Both vaccines will be injected into the amnion of chicken embryos. The tetradecylmaltoside surfactant will be evaluated as a gene delivery enhancer for both classes of vaccines. Hemagglutination- inhibition (HI) antibody titers against the A/Turkey/Wisconsin/68 virus will be analyzed in chickens post-hatch. Ad vectors will be generated using a new AdHigh system developed for manufacturing replication-competent Ad (RCA)-free Ad vectors at high speed and high titer. The overall goal of these experiments is to determine whether high HI antibody titers against avian influenza virus can be elicited in chickens by in ovo injection of non-replicating vectored vaccines without using the avian influenza virus itself, and thus avoiding the related biohazard of growing a deadly virus strain as well as difficulty of distinguishing the effect of vaccination from that of infection. [unreadable] [unreadable]